DE69528065T2 - AZAZYKLOHEXAPEPTID CONNECTIONS - Google Patents

Abstract

Compounds represented by the formula (Seq ID Nos. 1-6) <IMAGE> (I) wherein all substituents are fully defined, are disclosed. These compounds exhibit utilities as antibiotic and antifungal agents and for the treatment and prevention of Pneumocystis infections.

Description

BACKGROUND OF THE INVENTION

The present invention relates to azacyclohexapeptide compounds which may be useful as antifungal and anti-Pneumocystis agents.

There is currently a need for antifungal and anti-Pneumocystis agents due to an increase in the number of isolates resistant to conventional agents. In addition, conventional agents show somewhat high toxicity levels that limit their suitability. Finally, the incidence of Pneumocystis carinii pneumonia is increasing, especially given the susceptibility to infection of immunocompromised patients, such as those suffering from AIDS.

SUMMARY OF THE INVENTION

The compound of the present invention, Compound I (SEQ ID NO:1-6), is characterized by having a nitrogen atom bonded to the cyclohexapeptide ring at the 5-carbon atom of the 4-hydroxyornithine moiety (hereinafter "C-5-orn") and a hydroxy group bonded to the 4-position of the 5-membered ring of the proline moiety. The compound can be represented by the formula (I):

where

R&sub1; CH2 CH2 NH2 , CH2 CN or CH2 CONH2 is,

R₂ is H or OH,

RI is C9 -C21 alkyl, C9 -C21 alkenyl,

RII H, C1 -C4 alkyl, C3 -C4 alkenyl, (CH2 )2-4 OH, (CH2 )2-4 NRIVRV, CO(CH2 )1 -4 NH2 is,

RIII is H, C1 -C4 alkyl, C3 -C4 alkenyl, (CH2 )2-4 OH, (CH2 )2-4 NRIVRV,

RIV is H or C₁-C₄-alkyl,

RV is H or C₁-C₄-alkyl,

or a pharmaceutically acceptable acid addition salt and/or hydrate thereof.

Particularly preferred is the compound wherein R1 is CH2CH2NH2, R2 is OH, RI is 9,11-dimethyltridecyl, RII is CH2CH2NH2 and RIII is hydrogen.

In the description and the appended claims, a chemical formula or chemical name is intended to encompass all optical and stereoisomers as well as racemic mixtures, where such isomers and mixtures exist.

The term alkyl means hydrocarbon groups with a straight, branched or cyclic chain, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl, cyclohexylmethyl and the like.

The term cycloalkyl means an alkyl species with 3 to 15 carbon atoms without alternating or mesomeric double bonds between the carbon atoms.

The term alkenyl means groups such as vinyl, 1-propen-2-yl, 1-buten-4-yl, 2-buten-4-yl, 1-penten-5-yl and the like.

The term alkoxy means straight- or branched-chain oxyalkyl groups, such as methoxy, ethoxy, butoxy, heptoxy, dodecyloxy and the like.

Pharmaceutically acceptable salts suitable as acid addition salts include salts of inorganic acids such as hydrochloric, hydrobromic, sulphuric, nitric, phosphonic and perchloric acid, and salts of organic acids such as tartaric, citric, acetic, succinic, maleic, fumaric and oxalic acid, as well as other essential non-toxic acid addition salts known to those skilled in the art.

Representative cores for the aza derivatives of the present invention (Compound I) and the sequence identification for these compounds can be found in the following table. Since the peptide cores would be the same regardless of the substituents RI, RII or RIII and since the sequence identification number is assigned to the core variations, the amines and salts have the same sequence identification numbers.

The compounds are soluble in lower alcohols and in polar aprotic solvents such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and pyridine. They are insoluble in solvents such as diethyl ether and acetonitrile.

The compounds of the present invention are useful as an antibiotic, in particular as an antifungal agent or as an antiprotozoal agent. As antifungal agents they are useful for combating both filamentous fungi and yeasts. They are particularly useful for treating mycotic infections in mammals, especially those caused by Candida species such as C. albicans, C. tropicalis and C. pseudotropicalis, Cryptococcus species such as C. neoformans and Aspergillus species such as A. fumigatus, A. flavus, A. niger. They are also useful for treating and/or preventing Pneumocystis carinii pneumonia, to which immunocompromised patients are particularly susceptible, as described hereinafter.

The compounds of the present invention can be obtained from Connection with the formula

by a series of reactions in which the oxygen atom at the "C-5 orn" (or hemiaminal) position is ultimately substituted by nitrogen. The starting material (Compound A) for preparation can be a natural product in which R¹ is 9,11-dimethyltridecyl and can be produced by culturing Zalerion arbicola ATCC 20868 in a nutrient medium enriched in mannitol as the main carbon source as described in the US patent.

The sequence identification numbers of the starting materials are shown in the following table:

The reaction sequence is shown in the scheme below.

* The position is the "C-5-orn" or hemiaminal position.

In step A, the starting material compound A (SEQ ID NO:7-12), alkylthiol or arylthiol and acid are reacted in an aprotic solvent under anhydrous conditions for a time sufficient for the reaction to proceed to form compound B (SEQ ID NO:13-18) as shown in the following table. Aminoethylthiol has been found to be suitable for this step.

For step A, suitable acids include strong organic acids and mineral acids. Examples of strong organic acids include camphorsulfonic acid, p-toluenesulfonic acid and methanesulfonic acid. Mineral acids include hydrochloric acid and hydrobromic acid. Camphorsulfonic acid is preferred.

Suitable solvents include DMF, DMSO, 1-methyl-2-pyrrolidinone and hexamethylphosphoric triamide (HMPA). DMF or DMSO is preferred.

The reaction is generally carried out at ambient temperature for 1 to about 10 days.

In carrying out the reaction, the cyclohexapeptide compound, the thiol compound and the acid are stirred together in a suitable solvent until the reaction is substantially complete. The reaction mixture is then diluted with water and flash chromatographed on reverse phase resins using 10 to 40 percent acetonitrile/water (containing 0.1% trifluoroacetic acid) as the eluent. Trifluoroacetic acid may be referred to hereinafter as "TFA". Fractions containing the desired product may be concentrated and freeze dried and the freeze dried material purified by preparative high performance liquid chromatography (HPLC).

Suitable HPLC columns are commercially available columns sold under brand names or trade names such as "ZORBAX" (DuPont), "DeltaPak" (Waters), Bio-Rad (Bio-Rad), "LICHROPREP"RP18 (E. Merck). The specific columns are indicated in the working examples.

In step B, compound C (SEQ ID NO:13-18), a sulfone, is obtained by the oxidation of compound B. Suitable oxidizing agents or oxidants include "OXONE" (KHSO5·KHSO4·K2SO4 2:1:1, Aldrich Chemicals), metachloroperoxybenzoic acid, and peroxyacetic acid. The sequence identification number of compound C is the same as that of compound B because the atom attached to the hemiaminal carbon is still sulfur. Thus, the sequence identification numbers of the sulfones are as follows:

The oxidation of the thioether (Compound B) to the sulfone (Compound C) is carried out with approximately two molar amounts of the oxidizing agent. If a one molar amount of oxidizing agent is used, the product is a sulfoxide, which can then be converted to the sulfone. The sulfoxides can be used as an intermediate in the formation of the aza compounds, but the sulfone is preferred. A slight excess over the two molar amount of the oxidizing agent is used.

The reaction is carried out in an aqueous medium, preferably in a mixture of acetonitrile and water. Approximately equal amounts are preferred, although a range of 1:9 to 9:1 may be employed.

When carrying out the reaction, the oxidizing agent is added to a solution of compound B (Seq. Ident. No. 13-18) in 1 : 1 Acetonitrile/water is added and the mixture is allowed to stand at ambient temperature for a time sufficient to complete the reaction, generally about 30 minutes to one hour, to obtain compound C.

After the reaction is complete, the compound is recovered from the reaction mixture by dilution with water and chromatography. Reverse phase (C18) flash column chromatography is suitable for this purification step. The preferred eluent is 30-45 percent acetonitrile/water (0.1% TFA) with a gradient in 5 percent steps. The appropriate fractions are freeze dried to obtain the desired sulfone intermediate, compound C (SEQ ID NO: 13-18). The intermediate tends to be unstable, so isolation should be carried out as quickly as possible.

Compound C can be converted to a compound having a nitrogen atom directly attached to the "C-5-orn". As can be seen in the flow diagram, reaction of Compound C with an alkali metal azide produces an azide at that position (Compound D), while reaction with an amine compound (ammonia or amine) produces an amino group at the "C-5-orn" position (Compound I). Compound D is an important intermediate for most of the compounds of the present invention. Although Compound D has a nitrogen atom at "C-5-orn", since Compound D is not a product, separate sequence identification numbers are assigned. The sequence identification numbers for Compound D can be found in the table below.

The azide can be obtained by adding alkali metal azide to a solution of the sulfone (Compound C, SEQ ID NO:13-18) in an aprotic solvent with stirring at ambient temperature for a time sufficient to complete the reaction to form the azide as determined by HPLC analysis. The reaction mixture can then be diluted with aqueous acid such as trifluoroacetic acid and then chromatographed to separate the desired azide (Compound D) from the reaction mixture. Reverse phase (C18) flash column chromatography using 10-25 percent acetonitrile/water (0.1% TFA) with a gradient in 5 percent increments is suitable for this procedure.

The azide (Compound D) can then be reduced to a compound having a free amino group, which is among the products (Compound I, SEQ ID NO:1-6) of the present invention.

The reduction can be carried out by mixing the azide compound (Compound I) with Pd/C in a solvent such as glacial acetic acid and hydrogenating under balloon pressure for 10 to 20 hours. The product can then be recovered by first removing the catalyst by filtration and freeze-drying the filtrate to obtain the amine compound (SEQ ID NO: 1-6) in which the amine is a primary amine.

The resulting amine can be converted into a substituted amine as described below.

Compound I, wherein -NRIIRIII is represented by -NHCH2CH2NH2 or generally by -NH(CH2)2-4NRIVRV, can be prepared from the sulfone by a process in which a diamine H2N(CH2)2-4NRIVRV is reacted with the sulfone (Compound C, SEQ ID NO: 13-18).

The reaction is carried out in an aprotic solvent, such as one of those mentioned above, and at ambient temperature. About a 10-fold molar excess of the amine compound is used. The reaction can be carried out for one or more hours.

When carrying out the reaction, the appropriate amine is added to a solution of the sulfone in anhydrous aprotic solvent and the reaction mixture stirred at ambient temperature to give compound I (SEQ ID NO:1-6) wherein the substituent at "C-5-orn" is -NRIIRIII. The desired compound can then be recovered by dilution with aqueous trifluoroacetic acid followed by chromatography. Reverse phase (C18) flash column chromatography using 10 to 25% acetonitrile/water (0.1% TFA) as eluent with a gradient in 5% steps is suitable. The appropriate fractions can be freeze dried to recover the product as a trifluoroacetate salt.

The trifluoroacetate salt can be converted by dissolving the salt in water and passing it through a Bio-Rad AG2-X8(Cl-) Polyprep column and recovering the product as the hydrochloride salt.

The amines prepared as above and having a described primary amino group -NR2 can then be alkylated by conventional means to obtain a substituted amino group. Briefly, the alkylation can be carried out by reacting an appropriately substituted alkyl halide with the amine (Compound I, NRIIRIII=NH2; SEQ ID NO:1-6) in an aprotic solvent in the presence of a base to obtain the monosubstituted amine (Compound I, NRIIRIII=NHRII, where RII is C1-C4 alkyl, C3-C4 alkenyl, (CH2)2-4 OH and (CH2)2-4 NRIVRV). The latter can be obtained from the reaction mixture by conventional methods.

The amines prepared as described above and having a primary amino group -NH₂ can be acylated by conventional means to obtain an acylated amino group. The desired acyl group is CO(CH₂)₁₋₄NH₂. Since this is a primary amino group, the amino moiety of the acylating acid is protected before carrying out the acylation, e.g. with a benzyloxycarbonyl group. Preferably an activated ester such as the pentafluorophenyl ester is used. The acylation can be carried out in an aprotic solvent in the presence of a base such as diisopropylethylamine at ambient temperature for one or more hours to obtain the acylation product. The product can be recovered by diluting the reaction mixture with methanol and purifying by HPLC. The protecting group can be removed by conventional hydrogenolysis. (Compound I, -NRIIRIII=-NHCO(CH₂)₁₋₄NH₂).

The amine compounds in which the amino group is completely substituted in the hemiaminal position, ie when neither RII nor RIII in

is hydrogen are preferably prepared by reacting the sulfone (Compound B, SEQ ID NO: 19-24) with an appropriately substituted amine RIIRIIINH. The reaction can be carried out by adding the amine to a stirred solution of the sulfone for a time sufficient for the reaction to take place. The product can be recovered by purification by preparative HPLC and freeze-drying the appropriate components.

The invention also includes acid addition salts. The compound is obtained in the normal course of isolation as an acid addition salt. Generally it is present as a trifluoroacetic acid salt. The salt thus obtained can be dissolved in water and passed through an anion exchange column bearing the desired anion. The eluate containing the desired salt can be concentrated to recover the salt as a solid product.

The compounds of the present invention are active against many fungi and in particular against Candida species. The antifungal properties can be illustrated by determining the minimum fungicidal concentration (MFC) against certain Candida organisms in a micronutrient solution dilution assay performed in yeast nitrogen base (Difco) medium with 1% dextrose (YNBD).

In a representative study, the compounds are dissolved in 100% dimethyl sulfoxide (DMSO) with an initial concentration of 5 mg/ml. After dissolution, the drug stock solution is diluted in water to a concentration of 512 ug/ml so that the final DMSO concentration was approximately 10 percent. The solution is then dispensed using a multichannel pipettor into the first row of a 96-well plate (each well containing 0.075 ml of YNBD) to give a drug concentration of 256 ug/ml. The compounds in the first row are diluted 2-fold across the rows to give final drug concentrations ranging from 256 ug/ml to 0.12 ug/ml.

Four-hour broth cultures of organisms to be tested are calibrated using a spectrophotometer at 600 nm to match a 0.5 MeFarland standard. This suspension is diluted 1:100 in YNBD to give a cell concentration of 1-5 x 104 colony forming units (CFU)/ml. Aliquots of the suspension (0.075 ml) are inoculated into each well of the microtiter plate, giving a final cell inoculation of 5-25 x 103 CFU/ml and final drug concentrations ranging from 128 ug/ml to 0.06 ug/ml. Each assay includes one row for drug-free control wells and one row for cell-free control wells.

After 24 hours of incubation, the microtiter plates are gently shaken on a shaker to resuspend the cells. The MIC-2000 inoculator is used to transfer a 1.5 microliter sample from each well of the 96-well microtiter plate to a single well inoculum plate containing Sabouraud dextrose agar (SDA). The inoculated SDA plates are incubated at 35ºC for 24 hours and then read to determine the minimum fungicide concentration (MFC). The MFC is defined as the lowest drug concentration at which no growth or less than 4 colonies per spot occurs.

The in vivo antifungal activity of the compounds can be demonstrated by the following study.

The overnight growth of an SDA culture of C. albicans MY 1055 is suspended in sterile saline and the cell concentration by hemocytometer counting and the cell suspension is adjusted to 3.75 x 10⁵ cells/ml. Then 0.2 milliliters of this suspension is administered intravenously into the tail vein of mice so that the final inoculation is 7.5 x 10⁴ cells/mouse.

The study is then performed by intraperitoneal (i.p.) administration of aqueous solutions of Compound I at various concentrations, twice a day (BID), for four consecutive days, to 18-20 gram female DBA/2 mice previously infected with C. albicans as described above. As a control, distilled water is administered i.p. to mice infected with C. albicans. After seven days, the mice are sacrificed by carbon dioxide gas, the paired kidneys are aseptically removed and placed in sterile polyethylene bags containing 5 milliliters of sterile saline. The kidneys are homogenized in the bags, serially diluted in sterile saline and aliquots spread on the surface of SDA plates. Plates are incubated at 35ºC for 48 hours and yeast colonies are counted to determine colony forming units (CFU) per gram of kidney.

The compounds of the present invention may also be useful for inhibiting or alleviating Pneumocystis carinii infections in immunocompromised patients. The efficacy of the compounds of the present invention for therapeutic or anti-infective purposes can be illustrated by studies in immunocompromised rats.

Sprague-Dawley rats (weighing approximately 250 grams) are immunosuppressed with dexamethasone in the drinking water (2.0 mg/L) and maintained on a low-protein diet for seven weeks to stimulate the development of Pneumocystis pneumonia from latent infection. Before drug treatment, rats are sacrificed to confirm the presence of Pneumocystis carinii pneumonia (PCP). Five rats (weighing approximately 150 grams) are injected subcutaneously (sc) with Compound I in 0.25 ml of vehicle (distilled water) twice daily for four days. A control experiment with vehicle is also performed. All animals continued to receive dexamethasone in drinking water and a low-protein diet during the treatment period. After treatment was terminated, all animals were sacrificed, the lungs removed and processed, and the extent of disease was determined by microscopic analysis of stained sections. Prevention or reduction of cysts was evident in sections of the lungs of treated rats when compared to the number of cysts in the lungs of untreated or solvent controls.

The excellent properties are most effectively utilized when the compound is formulated into new pharmaceutical compositions with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding procedures.

The novel compositions contain at least a therapeutic antifungal or antipneumocystic amount of the active compound. Generally, the composition contains at least 1% by weight of Compound I. Concentrate compositions suitable for dilution prior to use may contain 90% by weight or more. The compositions include compositions suitable for oral, topical, parenteral (including intraperitoneal, subcutaneous, intramuscular and intravenous), nasal and suppository administration, or for insufflation. The compositions may be prepackaged by intimately mixing Compound I with the components appropriate for the desired medium.

Compositions formulated for oral administration may be liquid or solid compositions. In liquid preparations, the therapeutic agent may be formulated with liquid carriers such as water, glycols, oils, alcohols and the like, and in solid preparations such as capsules and tablets, with solid carriers such as starches, sugars, kaolin, ethylcellulose, calcium and sodium carbonate, calcium phosphate, kaolin, talc, lactose, generally with lubricants such as calcium stearate, together with binding agents, disintegrants and the like. Because of their ease of administration, tablets and Capsules represent the most advantageous oral dosage form. It is particularly advantageous to formulate the compositions in unit dosage form (defined hereinafter) for ease of administration and uniform dosing. Unit dosage compositions form one aspect of the present invention.

The compositions may be formulated for injection and may take forms such as suspensions, solutions or emulsions in oily or aqueous vehicles such as 0.85 percent sodium chloride or 5 percent dextrose in water and may contain formulators such as suspending, stabilizing and/or dispersing agents. Buffering agents as well as additives such as saline or glucose may be added to make the solutions isotonic. The compound may also be dissolved in alcohol/propylene glycol or polyethylene glycol for intravenous drip administration. These compositions may also be presented in unit dose form in ampoules or in multidose containers, preferably with an attached preservative. Alternatively, the active ingredients may be in powder form for reconstitution with a suitable vehicle prior to administration.

The term "unit dosage form" as used in the specification and claims refers to physically discrete units, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier. Examples of such unit dosage forms are tablets, capsules, pills, powder packets, wafers, metered units in ampoules or in multidose containers, and the like. A unit dosage form of the present invention will generally contain from 100 to 200 milligrams of one of the compounds.

If the compound is intended for antifungal purposes, any route of administration may be used. For the treatment of mycotic infections, oral or intravenous administration is usually used.

If the compound to combat Pneumocystis infections is used, it is desirable to treat the lungs and bronchi directly. For this reason, inhalants are preferred. For administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or nebulizers. The preferred delivery system for inhalation is a metered dose inhalation aerosol (MDI) which may be formulated as a suspension or solution of compound I or II in suitable propellants such as fluorocarbons or hydrocarbons.

Although the compounds of the present invention can be employed as tablets, capsules, topical compositions, insufflation powders, suppositories and the like, the solubility of the compounds of the present invention in water and aqueous media makes them suitable for use in injectable formulations and also in liquid compositions suitable for aerosol sprays.

The following examples illustrate the invention, but are not to be construed as limiting. EXAMPLE 1

Part A Preparation of Aminoethyl Thioether Intermediate (SEQ ID NO: 15)

A solution of 500 mg (0.47 mmol) of 1-[4,5-dihydroxy-N2-(10,12- dimethyl-1-oxotetradecyl)ornithine]-5-(3-hydroxyglutamine)-6-(4-hydroxyproline)echinocandin B, 5.34 grams (47 mmol) of 2-aminoethanethiol hydrochloride, and 109 mg (0.47 mmol) of (1S)-(+ )-10-camphorsulfonic acid in 40 mL of anhydrous N,N-dimethylformamide is stirred at 25C for 2-6 days or a time sufficient to cause the disappearance of the starting material. The reaction is diluted with 40 mL of water and flash chromatographed on "LICHROPREP" (E. Merck) RP 18 (40-63 µm, 15 grams) packed with 10% acetonitrile/water. The column is eluted with 10 to 40% acetonitrile/water, collecting two 120 mL fractions at each gradient step. The appropriate fractions, as determined by analytical HPLC (Zorbax Rx C18, 40% acetonitrile/water/0.1% trifluoroacetic acid, 210 nm), are concentrated and lyophilized. The residue is further purified by preparative HPLC (ZORBAX C18, 40% acetonitrile/water/0.1% TFA, 210 nm) to give the desired compound as a trifluoroacetate salt with a molecular weight of 1238.

Part B Oxidation to sulfone (SEQ ID NO: 15)

The mixture of thioethers obtained as described above (0.358 mmol) is dissolved in 15 mL of 1:1 acetonitrile/water and "OXONE" (1.06 mmol equivalents of potassium hydrogen persulfate) is added. After about one hour, the solution is diluted with an equal volume of water and rapidly chromatographed using reverse phase C18 flash chromatography with 35-45% acetonitrile/water containing 0.1% TFA in 2% step gradients as eluant. The product-containing fractions are freeze dried to give the product with a molecular weight of 1270.

Part C Replacement of sulfones by azide (SEQ ID NO: 21)

The mixture of sulfones (0.257 mmol) prepared as described above is dissolved in 10 mL of anhydrous DMF. Lithium azide (0.257 mmol) is added as a solid and the mixture is stirred for about Stirred for 4-24 hours. The mixture is purified by reverse phase C18 flash chromatography using 30-65% acetonitrile/water in 5% step gradients as eluent. The appropriate fractions as determined by reverse phase HPLC (RP-18, 40% acetonitrile/water/0.1% TFA, 210 nm) are collected, frozen and freeze dried to give the crude product. Further purification by preparative reverse phase HPLC (C18, 40-45% acetonitrile/water/0.1% TFA, 210 nm) gives the desired purified compound with a molecular weight of 1090.

Part D Reduction of the azide to the amine (SEQ ID NO: 3)

A mixture of the azide compound (0.126 mmol) (obtained as described above) and 10% Pd on carbon (100-150 mg) is suspended in glacial acetic acid (10 mL). The reaction vessel is purged first with nitrogen, then with hydrogen. One atmosphere of hydrogen gas pressure is maintained for a time sufficient to give complete reduction to the amine product, typically 2 to 24 hours. The catalyst is removed by filtration and the filtrate is freeze-dried to give the crude amine. Further purification can be achieved by preparative reversed-phase chromatography (C18, 35-41% acetonitrile/water/0.1% TFA in 3% step gradients, 210 nm). The product-containing fractions are freeze-dried to give the purified compound with a molecular weight of 1178. EXAMPLE 2

The sulfone mixture (0.945 mmol) obtained as described above in Part B of Example 1 is dissolved in 20 mL of anhydrous DMF and ethylenediamine (9.45 mmol) is added. The mixture is stirred for approximately 1-12 hours or until analytical HPLC analysis (RP-18, 40% acetonitrile/water/0.1% TFA, 210 nm) indicates complete disappearance of the starting sulfone. The mixture is separated by reverse phase (C18) flash column chromatography using 10-40% acetonitrile/water/0.1% TFA in 5% step gradients as eluent. The appropriate fractions are collected, frozen and freeze dried to give the desired product with the α-C-5-orn configuration and its β-C-5-orn epimer. The resulting trifluoroacetate salt is dissolved in a small volume of deionized water and passed through a Bio-Rad AG2-X8 (Cl-) polyprep column, washing with additional water. The product-containing eluate is freeze-dried to give the desired product as the dihydrochloride salt with a molecular weight of 1180. EXAMPLE 3

The sulfone mixture (0.945 mmol) obtained as described in Part B of Example 1 is dissolved in 20 mL of anhydrous DMF and n-butylamine (9.45 mmol) is added. The mixture is stirred for approximately 1-12 hours or until analytical HPLC analysis (RP-18, 40% acetonitrile/water/0.1% TFA, 210 nm) indicates complete disappearance of the starting sulfone. The mixture is separated by reversed-phase (C18) flash column chromatography using 10-50% acetonitrile/water/0.1% TFA in 5% step gradients as eluent. The appropriate fractions are collected, frozen and freeze-dried to give the desired product with the α-C-5-orn configuration and its β-C-5-orn epimer. The products, isolated as their trifluoroacetate salts, have a molecular weight of 1234. EXAMPLE 4

The sulfone mixture (0.945 mmol) obtained as described in Part B of Example 1 is dissolved in 20 mL of anhydrous DMF and ethanolamine (9.45 mmol) is added. The mixture is stirred for approximately 1-12 hours or until analytical HPLC analysis (RP-18, 40% acetonitrile/water/0.1% TFA, 210 nm) indicates complete disappearance of the starting sulfone. The mixture is separated by reverse phase (C18) flash column chromatography using 10-40% acetonitrile/water/0.1% TFA in 5% step gradients as eluent. The appropriate fractions are collected, frozen and freeze dried to give the desired product with the α-C-5-orn configuration and a molecular weight of 1222. EXAMPLE 5

Part A Preparation of the intermediate nitrile compound (SEQ ID NO: 2)

A solution of 250 milligrams (0.23 mmol) of 1-[4,5-dihydroxy- N2-10,12-dimethyl-1-oxotetradecyl)ornithine]-5-(3-hydroxyglutamine)- 6-(4-hydroxyproline)echinocandin B was prepared in 3.0 mL of N,N-dimethylformamide. 64 milligrams of cyanuric chloride was added in one portion and the mixture was stirred for 5.5 minutes and immediately quenched with 0.55 mL of 2 M sodium acetate solution. The mixture was purified by preparative HPLC ("ZORBAX" C8, acetonitrile/water/0.1% TFA, 210 and 277 nm). The appropriate product-containing fractions as determined by analytical HPLC ("ZORBAX" C8, 45% water/acetonitrile/0.1% TFA, 1.5 mL/minute, 210 and 277 nm) were pooled and freeze-dried to yield 45 mg of the desired nitrile compound >98% pure as a white solid. 1H NMR (400 MHz, CD3OD) δ 7.12 (d, 1H), 6.74 (d, 1H), 5.31 (m, 1H), 2.83 (dd, 1H), 2.72 (dd, 1H), 2.42 (m, 1H), 1.21 (d, 3H); FAB-MS (Li), m/e 1054 (M + H + Li)

Part B Reduction of the nitrile to the amine (SEQ ID NO: 1)

To a solution of 38.5 mg (0.036 mmol) of the nitrile (prepared in Part A above) in 2.0 mL of methanol was added 32 mg (0.25 mmol) of CoCl2·H2O. Then 46 mg (34 equivalents) of sodium borohydride was added in several portions. The mixture was stirred under a nitrogen atmosphere at room temperature for 3 h. The reaction mixture was purified by preparative HPLC ("ZORBAX" C8, 45% water/acetonitrile, 210 and 277 nm). The appropriate fractions as determined by analytical HPLC ("ZORBAX" C8, 45% water/acetonitrile/0.1% TFA, 1.5 mL/minute, 210 and 277 nm) were pooled and freeze-dried to give 13 mg of the trifluoroacetate salt of the desired amine (> 98% pure) as a white solid. FAB-MS (Li), m/e 1058 (M + H + Li).

Part C Preparation of the aminoethyl thioether (SEQ ID NO: 13)

A solution of the amine prepared in Part B above (0.047 mmol), 2-aminoethanethiol hydrochloride (4.7 mmol), and (15)- (+)-10-camphorsulfonic acid (0.047 mmol) in 4 milliliters of anhydrous N,N-dimethylformamide is stirred at 25°C for 2-6 days or a time sufficient to cause the starting material to disappear. The reaction is diluted with 4 mL of water and flash chromatographed on "LICHROPREP" (E. Merck) RP 18 (40-63 µm, 1.5 grams) packed with 10% acetonitrile/water. The column is eluted with 10 to 40% acetonitrile/water, collecting two 12 mL fractions at each gradient step. The appropriate fractions, as determined by analytical HPLC (Zorbax Rx C18, 30% acetonitrile/water/0.1% trifluoroacetic acid, 210 nm), are concentrated and freeze-dried. The residue is further purified by preparative HPLC (ZORBAX C18, 30% acetonitrile/water/0.1% TFA, 210 nm) to give the desired isomeric compounds as ditrifluoroacetate salts, both of which have a molecular weight of 1338.

Step D Oxidation to sulfone (SEQ ID NO: 13)

The mixture of thioethers obtained as described above (0.036 mmol) is dissolved in 1.5 mL of 1:1 acetonitrile/water and "OXONE" (0.106 mmol equivalents of potassium hydrogen persulfate) is added. After about one hour, the solution is diluted with an equal volume of water and rapidly chromatographed using reversed phase C18 flash chromatography with 35-45% acetonitrile/water containing 0.1% TFA in 2% step gradients as eluent. The product-containing fractions are freeze-dried to give the product of molecular weight 1370.

Part E Replacement of sulfones by ethylenediamine (SEQ ID NO: 1)

The mixture of sulfones (0.094 mmol) obtained as described above in Part D is dissolved in 2.0 mL of anhydrous DMF and ethylenediamine (0.94 mmol) is added. The mixture is stirred for approximately 1-12 hours or until analytical HPLC analysis (RP-18, 30% acetonitrile/water/0.1% TFA, 210 nm) indicates complete disappearance of the starting sulfone. The mixture is separated by reversed-phase (C18) flash column chromatography using 10-30% acetonitrile/water in 5% step gradients as eluent. The appropriate fractions are collected, frozen and freeze-dried to give the desired product with the α-C-5-orn configuration and its β-C-5-orn epimer. The resulting trifluoroacetate salt is dissolved in a small volume of deionized water and passed through a Bio-Rad AG2-X8 (Cl-) polyprep column, washing with additional water. The product-containing eluate is freeze-dried to give the desired product as the dihydrochloride salt with a molecular weight of 1166. EXAMPLE 6

Part A Acylation with protected glycine

The amino compound obtained in Example 1, Part D (0.10 mmol) is dissolved in 1 mL of anhydrous N,N-dimethylformamide under a nitrogen atmosphere. Diisopropylethylamine (0.11 mmol) and N-carbobenzyloxyglycine pentafluorophenyl ester (0.15 mmol) are added and the reaction is stirred at room temperature for 1-12 hours or until analysis by analytical HPLC ("ZORBAX" C18, 50% acetonitrile/water/0.1% TFA, 210 and 277 nm) indicates the end of the reaction. The mixture is diluted with 1 mL of methanol and purified by preparative HPLC ("ZORBAX" C18, 70% water/acetonitrile/0.1% TFA to 50% water/acetonitrile/0.1% TFA, 2-step gradient, 210 and 277 nm) to give the desired glycylated compound with a molecular weight of 1255.

Part B Hydrogenolysis of carbobenzyloxy-protected glycine compound

The pure carbobenzyloxy-protected Compound (0.075 mmol) is dissolved in a mixture of 3 mL of methanol, 1 mL of water and 0.2 mL of glacial acetic acid. Then 50 mg of 10% palladium on carbon is added and the reaction vessel is purged first with nitrogen and then with hydrogen. The reaction is stirred vigorously under 1 atmosphere of hydrogen for several hours. The catalyst is removed by filtration and the volatiles are removed by rotary evaporation under reduced pressure. The residue is dissolved in 2 mL of water, frozen and freeze dried to give the desired deprotected amine product as a solid. The desired product has a molecular weight of 1181. EXAMPLE 7

Part A Replacement of sulfones by azide (Seq. Ident. No. 19)

The mixture of sulfones (0.257 mmol) prepared as described in Part D of Example 5 is dissolved in 10 mL of anhydrous DMF. Lithium azide (0.257 mmol) is added as a solid and the mixture is stirred for approximately 4-24 hours. The mixture is purified by reversed-phase C18 flash chromatography using 20-55% acetonitrile/water in 5% step gradients as eluent. The appropriate fractions as determined by reversed-phase HPLC (RP-18, 40% acetonitrile/water/0.1% TFA, 210 nm) are collected, frozen and freeze-dried to give the crude product. Further purification by preparative reversed-phase HPLC (C18, 30-40% acetonitrile/water/0.1% TFA, 210 nm) gives the desired purified compound with a molecular weight of 1190.

Part B Reduction of the azide to the amine (SEQ ID NO: 1)

A mixture of the azide compound (0.126 mmol) (obtained as described above in Part A) and 10% Pd on carbon (100-150 mg) is suspended in glacial acetic acid (10 mL). The reaction vessel is purged first with nitrogen, then with hydrogen. One atmosphere of hydrogen gas pressure is maintained for a time sufficient to give complete reduction to the amine product, typically 2 to 24 hours. The catalyst is removed by filtration and the filtrate freeze dried to give the desired amine as a diacetate salt with a molecular weight of 1170. EXAMPLE 8

The sulfone mixture (0.094 mmol) obtained as described above in Part D of Example 5 is dissolved in 2.0 mL of anhydrous DMF and N-methyl-N-allylamine (0.945 mmol) is added. The mixture is stirred for approximately 1-12 hours or until analytical HPLC analysis (RP-18, 40% acetonitrile/water/0.1% TFA, 210 nm) indicates complete disappearance of the starting sulfone. The mixture is separated by reverse phase (C18) flash column chromatography using 10-40% acetonitrile/water/0.1% TFA in 5% step gradients as eluent. The appropriate fractions are collected, frozen and freeze dried to give the desired product with the α-C-5-orn configuration and a molecular weight of 1332. EXAMPLE 9

The sulfone mixture (0.094 mmol) obtained as described above in Part D of Example 5 is dissolved in 2.0 mL of anhydrous DMF and N,N-dimethyl-1,3-diaminopropane (0.945 mmol) is added. The mixture is stirred for approximately 1-12 hours or until analytical HPLC analysis (RP-18, 40% acetonitrile/water/0.1% TFA, 210 nm) indicates complete disappearance of the starting sulfone. The The mixture is separated by reversed phase (C18) flash column chromatography using 10-40% acetonitrile/water/0.1% TFA in 5% step gradients as eluent. The appropriate fractions are collected, frozen and freeze dried to give the desired product with the α-C-5-orn configuration and a molecular weight of 1363.5.

The following examples illustrate representative compositions containing the compounds of the invention.

EXAMPLE A

1000 compressed tablets, each containing 500 mg of the compound of Example 5, are prepared from the following formulation:

Compound grams

Connection of Example 5 500

Strength 750

Dibasic aqueous calcium phosphate 5000

Calcium stearate 2.5

The finely powdered components are mixed well and granulated with 10 percent starch paste. The granulation is dried and pressed into tablets.

EXAMPLE B

1000 hard gelatin capsules, each containing 500 mg of the same compound, are prepared from the following formulation:

Compound grams

Connection of Example 5 500

Strength 250

Lactose 750

Talk 250

Calcium stearate 10

A uniform mixture of the ingredients is prepared by blending and used to fill two-piece hard gelatin capsules.

EXAMPLE C

An aerosol composition having the following formulation may be prepared:

Per canister

Compound of Example 5 24 mg

Lecithin NF liquid concentrate 1.2 mg

Trichlorofluoromethane, NF 4.026 g

Dichlorodifluoromethane, NF 12.15 g

EXAMPLE D

250 milliliters of an injectable solution with the following formulation can be prepared by conventional methods :

Dextrose 12.5g

Water 250ml

Compound of Example 5 400 mg

The components are mixed and then sterilized for use.

Production of raw materials:

The starting material, Compound A-3, SEQ ID NO:3, wherein R¹ is 9,11-dimethyltridecyl, can be prepared by culturing Zalerion arboricola ATCC 20868 in a nutrient medium containing mannitol as the primary carbon source, as described in the US patent.

Starting materials in which RI is a group different from that of the natural product can be obtained by deacylation of the lipophilic group of the natural product by subjecting the natural product to a deacylating enzyme in a nutrient medium until substantial deacylation occurs, the enzyme having first been obtained by culturing a microorganism from the family Pseudomondaceae or Actinoplanaceae as described in Experentia 34, 1670 (1978) or in U.S. Patent Application No. 4,293,482. The deacylated cyclopeptide is recovered and then acylated by mixing with a suitable active ester RCOZ, where Z is halogen, pentachlorophenoxy, pentafluorophenoxy, p-nitrophenoxy and the like, to obtain compound a having the desired acyl group.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: BALKOVEC, JAMES M

BOUFFARD, FRANCES A

HAMMOND, MILTON L

(ii) TITLE OF THE INVENTION: AZACYCLOHEXAPEPTIDE COMPOUNDS

(iii) NUMBER OF SEQUENCES: 24

(iv) CORRESPONDENCE ADDRESS:

(A) NAME: ELLIOTT KORSEN

(B) STREET: P. O. Box 2000, 126 E. LINCOLN AVE

(C) CITY: RAHWAY

(D) STATE: NJ

(E) COUNTRY: USA

(F) Postcode: 07065

(v) COMPUTER-READABLE FORM:

(A) TYPE OF MEDIUM: Floppy disk

(B) COMPUTER: IBM PC Compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT REGISTRATION DETAILS:

(A) APPLICATION NUMBER:

(B) SUBMISSION DATE:

(CC) CLASSIFICATION:

(viii) INFORMATION ABOUT PATENT ATTORNEY/AGENT:

(A) NAME: KORSEN, ELLIOTT

(B) REGISTRATION NUMBER: 32705

(C) REFERENCE NUMBER/SIGN: 19305

(ix) TELECOMMUNICATIONS INFORMATION:

(A) PHONE: 908-594-5493

(B) FAX: 908-594-4720

(2) INFORMATION FOR SEQ ID NO: 1

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ü) MOLECULAR TYPE: Peptide

(iii) HYPOTHETICALLY: NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1

(2) INFORMATION FOR SEQ ID NO: 2

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ü) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2

(2) INFORMATION FOR SEQ ID NO: 3

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3

(2) INFORMATION FOR SEQ ID NO: 4

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4

(2) INFORMATION FOR SEQ ID NO: 5

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5

(2) INFORMATION FOR SEQ ID NO: 6

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6

(2) INFORMATION FOR SEQ ID NO: 7

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids.

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7

(2) INFORMATION FOR SEQ ID NO: 8

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8

(2) INFORMATION FOR SEQ ID NO: 9

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9

(2) INFORMATION FOR SEQ ID NO: 10

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10

(2) INFORMATION FOR SEQ ID NO: 11

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11

(2) INFORMATION FOR SEQ ID NO: 12

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12

(2) INFORMATION FOR SEQ ID NO: 13

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13

(2) INFORMATION FOR SEQ ID NO: 14

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14

(2) INFORMATION FOR SEQ ID NO: 15

(i) SEQUENCE FEATURES:

(R) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRENGTH: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15

(2) INFORMATION FOR SEQ ID NO: 16

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16

(2) INFORMATION FOR SEQ ID NO: 17

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17

(2) INFORMATION FOR SEQ ID NO: 18

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids.

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18

(2) INFORMATION FOR SEQ ID NO: 19

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19

(2) INFORMATION FOR SEQ ID NO: 20

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20

(2) INFORMATION FOR SEQ ID NO: 21

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid.

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21

(2) INFORMATION FOR SEQ ID NO: 22

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22

(2) INFORMATION FOR SEQ ID NO: 23

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23

(2) INFORMATION FOR SEQ ID NO: 24

(i) SEQUENCE FEATURES:

(A) LENGTH: 6 amino acids

(B) TYPE: Amino acid

(C) STRANGNESS: unknown

(D) TOPOLOGY: ring-shaped

(ii) MOLECULAR TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24

Claims (7)

1. A compound with the formula (Seq. Ident. No. 1-6) where. R&sub1; CH2 CH2 NH2 , CH2 CN or CH2 CONH2 is, R₂ is H or OH, RI is C9 -C21 alkyl, C9 -C21 alkenyl, RII H, C1 -C4 alkyl, C3 -C4 alkenyl, (CH2 )2-4 OH, (CH2 )2-4 NRIVRV, CO(CH2 )1 -4 NH2 is, RIII is H, C1 -C4 alkyl, C3 -C4 alkenyl, (CH2 )2-4 OH, (CH2 )2-4 NRIVRV, RIV is H or C₁-C₄-alkyl RV is H or C₁-C₄-alkyl, or a pharmaceutically acceptable acid addition salt and/or hydrate thereof or, where appropriate, a geometric or optical isomer or a racemic mixture thereof. 2. The compound of formula (Seq. Ident. No. 1) as defined in claim 1 where R&sub1; CH2 CH2 NH2 is, R₂ is OH, RI is 9,11-dimethyltridecyl, RII is CH₂CH₂NH₂ and RIII is hydrogen, or a pharmaceutically acceptable acid addition salt thereof. 3. An antibiotic composition comprising an effective amount of a compound as defined in claim 1 in a pharmaceutically acceptable carrier. 4. A composition according to claim 3 in unit dosage form, wherein the compound is present in an amount of about 10 to 200 milligrams. 5. The use of a compound as defined in claim 1 for the manufacture of a medicament for treating a fungal infection. 6. The use of a compound as defined in claim 1 for the manufacture of a medicament for preventing Pneumocystis infection. 7. The use of a compound as defined in claim 1 for the manufacture of a medicament for the treatment of Pneumocystis carinii infections.